The invention relates to methods for measuring the mobility of mass-selected ion species in an ion mobility spectrometer (IMS) coupled to a mass spectrometer (MS). Ion mobility spectrometers, including those connected to mass spectrometers, are usually operated by injecting very short ion current pulses. The ions are continuously generated in an ion source and then admitted into the drift region of the spectrometer by a gating grid over a short time span. The time spans for the transmission are usually between 100 and 300 microseconds; recording of the spectrum takes approx. 30 milliseconds. This method only uses a maximum of about one percent of the ions produced in the ion source. The low degree of ion utilization produces relatively poor signal-to-noise ratios in the mobility spectra obtained, for which reason attempts have repeatedly been made to improve the degree of ion utilization (ion efficiency). An increase from one percent to about 50 percent would, in theory, increase the signal-to-noise ratio, and thus the sensitivity of the method as well, by a factor of seven.
Bipolar grids are usually used as gating grids to generate the short ion current pulses. The ions transmitted by the grid are then pulled through a collision gas in a drift region by an axial electric field, their drift velocity being determined by their “mobility”, which, as is well known, in turn depends on their charge, their mass, their collision cross-section, their ability to become polarized, and also their tendency to form complex ions with molecules from the collision gas.
“Ion species” here denotes ions of a substance in a given charge state. The term ion species, as used here, includes both monoisotopic ions and the ions of the isotope satellites, but not ions of the same substance in different charge states. The ion species can consist of molecular ions or pseudomolecular ions, dimer ions or multimer ions, and all types of fragment ions. Ion complexes that form all types of bonds with molecules or molecular fragments of other substances shall also be included. Pseudomolecular ions are protonated or deprotonated molecules whose mass deviates from that of the molecule because of the mass of the proton.
All ions with the same charge experience the same tractive force from the electric field, but this force manifests itself in different drift velocities through the collision gas for ions with different mobilities, i.e. different collision cross-sections and different masses. For lighter ions in the order of magnitude of the mass of the collision gas, it is mainly the “reduced mass” of the ions, with minor influence of their collision cross-section, which determines their mobility; for heavier ions from several hundred or thousand atomic mass units upwards it is the particular form of the molecules that is decisive, the collision cross-section being the significant factor in the mobility. The collision cross-section depends to a large extent on the folding state of the ion, but also on the number of atoms in the molecule, and thus implicitly on the mass. The implicit dependence is roughly proportional to the square of the third root of the mass. The mobilities of ions of the same charge but different isotopic composition differ only slightly and cannot be separated in current mobility spectrometers.
In the ion sources usually applied in IMS, several ion species are generally formed from the molecules of one substance, mostly differing by charge, although they can also be ions of dimers or complexes with water and collision gas. Every ion species has a characteristic mobility. At the end of the drift region, the incident ion current is usually measured at an ion detector, digitized and stored as a “mobility spectrum” in the form of a digitized sequence of measured values. An evaluation of this mobility spectrum provides information on the mobilities of the ions involved and thereby—in pure mobility spectrometers—information about the substances involved.
The switching operation of the bipolar grid serves as the start time for measuring the drift velocity of the different bunches of ions. As the ions drift, the diffusion of the ions in the forwards and backwards direction generates a diffusion profile for each bunch of ions with the same mobility. In sufficiently long drift regions, this produces ion signals with the familiar bell-shaped curves of the Gaussian distribution to a very good approximation. The drift velocity is determined from the measured drift time in the center of the bell-shaped curve and the known length of the drift region in the drift tube of the spectrometer.
As a rule, the width of the bell-shaped curve of the ion signals is predominantly determined by diffusion. This results in a diffusion-defined mobility resolution Rd, which is almost constant across the mobility spectrum and proportional to the root of the ion charge, the strength of the electric drawing field and the length of the drift region. A reasonably good mobility spectrometer has a mobility resolution of about Rd=20 for singly charged ions, and this can just about satisfactorily resolve two ion species whose mobility differs by 10 percent because their signals differ by two complete full widths at half-maximum. Good mobility spectrometers have mobility resolutions of Rd=50 to 80 and can separate ions with mobility differences of only four percent or less. Today's best mobility spectrometers, developed non-commercially in a specialized research institutes, have mobility resolutions of Rd=150, which is sufficient to recognize two ion species whose mobilities only differ by just one percent.
In the following, we will initially deal with the better ion efficiency in pure ion mobility spectrometers. These spectrometers are often miniaturized, with drift regions of ten centimeters at most, operated at atmospheric pressure, and usually used to measure pollutants in ambient air. The pollutants, more generally called “analyte substances” below, are usually ionized in ambient air drawn in at atmospheric pressure, namely by so-called “chemical ionization at atmospheric pressure” (APCI) in reactions with reactant ions by protonation or deprotonation, whereby dimeric ions and complexes with water and collision gas molecules are also formed in addition to monomeric pseudomolecular ions. The ratios of the individual ion species with respect to each other depend on the concentration of the analyte molecules in the collision gas.
Nitrogen or air is usually used as collision gas, in which evenly distributed traces of water vapor (usually in carefully controlled concentrations) are present. The reactant ions are usually generated by beta emitters, for example 63Ni, but corona discharges and other electron beam generators and UV lamps are also used for this purpose. The reactant ions are formed in a reaction chain, which starts with the production of primary nitrogen ions and finishes with a number of different water complex ions. These water complex ions bring about the actual chemical ionization of the analyte molecules.
As they drift through the collision gas of the drift region at atmospheric pressure, the ions continually experience a very quick succession of new attachments and losses of H2O water molecules and N2 nitrogen molecules. Statistically averaged, an analyte ion, whether it be a monomer or a dimer, thus contains a×H2O and b×N2, where a and b are generally average, non-integral fractions. These changes happen very quickly, and so the peaks of the mobility spectrum are hardly broadened. If the ions of such a peak are transferred from atmospheric pressure into a connected mass spectrometer, a momentary state is frozen, just like in a flash photograph, and the mass spectrum obtained contains the ions with various states of attachment, and thus very different masses, side by side.
The following section describes attempts to increase the degree of utilization of the ions. F. J. Knorr et al. (Anal. Chem. 1985, 57, 402; U.S. Pat. No. 4,633,083 A) have proposed a method which operates with an axial ion beam modulated by two control grids. The modulation function used is a square-wave function, i.e. an alternating complete closing and complete opening of the grid. This type of modulation will be called “binary”. The first control grid is positioned directly behind the ion source, the second directly in front of the ion detector. Synchronous modulation of both grids generates an interference value for the ion beam at which some ion species can pass through while others are kept back by the interference of their drift time with the phases of the grid modulation frequency. If this modulation frequency is altered, an interference spectrum (“interferogram”) can be recorded, which can be transformed by means of a Fourier transformation from the frequency domain of the interferogram into the time domain, and thus into a mobility spectrum. The method, called “Fourier Transform Ion Mobility Spectrometry” by its authors, provides a theoretical ion utilization ratio of 25 percent because the ion quantities are halved at each of the two grids. Expectations for this method, however, were not fulfilled as far as the increase in the signal-to-noise ratio is concerned, and the method has not yet gained acceptance. In order to produce clean interferograms with this method, the modulation frequency must practically not vary at all during the drift time of the ions from the first gating grid to the second gating grid or to the detector. This requires the modulation frequency to change slowly.
In the patent specification U.S. Pat. No. 5,719,392 (J. Franzen, 1995), the ion current of an ion mobility spectrometer is modulated in a binary fashion by the gating grid with a rectangular temporal Hadamard pattern, where both the pulse widths of the ion packages transmitted as well as their separations are statistically distributed. The ion utilization thus increases to 50 percent. The evaluation to obtain the mobility spectrum can be done either by using a cross-correlation of the detector current with the applied pattern, or by using Fourier or Hadamard transformations. Using the Fourier transformation even makes it possible to obtain an improved mobility resolution by a partial deconvolution with the apparatus function. It has become apparent, however, that this evaluation procedure using the Fourier transform does not operate stably for a noisy detector signal. The method has not yet been used.
In a very recent patent application DE 10 2008 015 000.2 (U. Renner), the ion current from the ion source is analog modulated by a steady modulation function, e.g. a sine function, with an instantaneous frequency which varies over a wide frequency range; and the resulting ion current signal at the detector is decoded again by an analysis of the correlation with the modulation function. This results in an exceptionally noise-free mobility spectrum with very good mobility resolution. The mobility spectrum has an almost unprecedented quality. The ion utilization is 50 percent. The modulation can be performed with the usual gating grids that are present in these spectrometers. The modulation function can preferably be a linear or nonlinear “chirp”, as it is known from ion cyclotron resonance mass spectrometry. Even though the improvement to the signal-to-noise ratio does not quite match the theoretical expectations, the quality of the results and the stability of the method outclass all other attempts to obtain a mobility spectrum with high ion utilization.
The above-mentioned mobility spectrometers all operate at atmospheric pressure. There is now an almost universally accepted method of coupling them to mass spectrometers, which uses a different pressure range for the mobility drift region. A pressure range of about 500 pascals is used; the drift region is increased to a length of between 40 centimeters and two meters or more; and the electric field strength is increased to 2,000 volts per meter or more. In this pressure range, the drifting ions appear to form scarcely any complexes with other substances, so the mobilities of the ion species can be measured without any interference. However, in the long drift regions, the ions also diffuse in a radial direction over wide sections, which means that quite large diameters must be chosen for these drift regions. There is substantial patent literature for these applications, but they all operate in the conventional way with short, individual ion pulses which are introduced into the drift region. The duty cycle for the ions generated in the ion source here also amounts to only between 0.5 and 1 percent. The ion sources used are mainly electrospray ion sources (ESI). The mobility analyses are aimed mainly at peptides, proteins or other biopolymers in order to identify the folding structures of these biopolymers and determine the parallel existence of different folding structures for otherwise identical ions of an ion species.
Only the patent A. V. Loboda, U.S. Pat. No. 6,744,043 B2 (2004) will be mentioned here for these low-pressure methods with coupling to mass spectrometry because it offers an interesting axial focusing of the drifting ions in the drift region, albeit this has already been described and claimed in principle in the patent specification Thomson et al. U.S. Pat. No. 5,847,386 (1998). The Loboda patent specification proposes an RF ion guide with radial collision focusing for the drift region, the ion guide being constructed as an RF multipole rod system or as a ring system.
The publication of Mikhail E. Belov et al., Analytical Chemistry, Vol. 79, No. 6, Mar. 15, 2007, 2451 (“Multiplexed Ion Mobility Spectrometry Orthogonal Time-of-Flight Mass Spectrometry”) is the first to couple a low-pressure ion mobility spectrometer with high ion utilization to a mass spectrometer. Here, ions are pulsed into the ion mobility drift region just as usual, but the short ion pulses of equal duration are repeated with high repetition rate at quasi-stochastic time intervals, which are relatively long compared to the pulse duration. The time intervals were selected according to simulation trials so that they were between at least ten times up to 70 times longer than the pulse duration. Since the pulse duration is relatively short compared to the time interval between pulses, this method does not have a high ion utilization by itself; this could, however, be achieved by collecting the ions in an ion storage device before they are pulsed into the mobility drift region and by using special measures to pulse them in with as few losses as possible. For a primary ion beam with constant ion current, the varying collecting times produced varying numbers of ions in the individual pulses, which had to be taken into account during the evaluation by a calibration curve. This makes an otherwise very interesting publication complicated and, particularly, means that the dynamic measuring range of the TOF-MS cannot be utilized fully because there is always a danger that the TOF ion detector will be oversaturated by pulses with too high an ion density. The overall ion utilization given in this publication was about 50 percent, although this can be increased in principle to over 90 percent. The theoretical increase of the ion currents in the ion pulses was up to 70 times the ion current from the ion source, and the measured increase was up to 50 times. The dynamic measuring range of present-day commercial mass spectrometers is adjusted to the maximum ion currents achievable with the ion sources used. Even if these ion currents are only achieved with optimum substance supply, for example in the maximum of substance peaks from liquid chromatographs, excessive increases of the ion current are disadvantageous because they can lead to an oversaturation of the ion detector.
Depending on the substance supply to the ion source, time-of-flight mass spectrometers with orthogonal ion injection (OTOF) often only detect a few tens or hundreds of ions in a single mass spectrum, which is recorded in about 100 microseconds, and so the signal noise in these individual spectra is extraordinarily high. Such individual mass spectra cannot be evaluated individually in practice. Only in rare cases are around a thousand ions recorded in an individual mass spectrum at maximum substance supply, which means the saturation limit of the ion detector is reached. Since such an OTOF in normal operation requires many thousands of ions for a mass spectrum which can be readily evaluated, regular practice is to add together at least around 200, usually even between 500 and 1,000 mass spectra, to form a sum mass spectrum which can be evaluated. In the publication of Belov et al., in order to retain the time resolution of about ten kilohertz for measurement of the individual mass spectra for the mobility determination, the ion mobility separation was repeated 1,000 times, and the corresponding individual mass spectra from the repeated measurements were added together. Since every ion mobility separation takes about 127 milliseconds, this took a total time of 127 seconds.
Mass spectrometers can only ever determine the ratio of the ion mass to the charge of the ion. In the following, the term “mass of an ion” or “ion mass” always refers to the ratio of the mass m to the number z of elementary charges on the ion, i.e. the mass-to-elementary charge ratio m/z. The quality of a mass spectrometer is essentially determined by the mass resolution in addition to other criteria. The mass resolution is defined as R=m/Δm, where R is the resolution, m the mass of an ion measured in units of the mass scale, and Δm the full width of the mass signal at half maximum, measured in the same mass units.